Oral submucous fibrosis (OSF) is a debilitating irreversible fibrotic condition of the oral cavity. It is characterized by inflammation and ultimately results in trismus. Patients face difficulty in speaking, swallowing and chewing due to restricted mouth opening (trismus). This disease is also categorized as an oral premalignant disorder (OPMD). Recent reports cite a conversion rate of 10% from OSF to oral squamous cell carcinoma (OSCC). Epidemiological studies and case reports over the years have correlated the habit of chewing areca nut (Areca catechu) to the manifestation of OSF. It is a major cause of concern in the South and South East Asian parts of the world where areca nut is cultivated and routinely consumed. There are an estimated 700 million areca nut chewers around the globe with 0.5% of the population in the Indian subcontinent being affected by OSF due to this habit. Previous studies have reported differential gene expression profile and up regulation of the pro-fibrotic transforming growth factor-β (TGF-β) pathway in OSF. However, detailed molecular mechanisms for the pathogenesis of this disease are still unclear despite our knowledge about the etiological agent (areca nut) responsible for its progression. Therefore, to gain insights into the etiopathogeneses of OSF, following objectives were undertaken:  To study the gene expression changes induced by areca nut and pro-fibrotic cytokine TGF-β in primary fibroblast cells, and their implications in OSF.  To elucidate the mechanism of TGF-β signal activation in epithelial cells by areca nut. Fibroblast cells are the effectors in all fibrotic disorders. Therefore, it is essential to study the response of this cell type in fibrosis. With prior knowledge of the activation of TGF-β pathway in OSF and the etiological agent of this disease being areca nut; we wanted to study the differential gene response of fibroblasts to these two agents. For this purpose, human primary gingival fibroblasts (hGF) were used as a model system to study the global gene expression profile regulated by areca nut and/or TGF-β. hGF cells were treated with sub-cytotoxic dose of areca nut (5 µg/ml) with and without TGF-β (5 ng/ml) for 72 hours and microarray was performed. The results revealed 4666 genes being differentially regulated by areca nut in hGF cells while TGF-β regulated 1214 genes. Both of them together differentially regulated 5752 genes. 413 genes which were commonly regulated by areca nut and TGF-β were observed to have enhanced regulation with a combined treatment of areca nut, together with TGF-β. This result pointed towards the potential role of both areca nut and TGF-β in modulating fibroblast response. To further assess the role of areca nut in OSF manifestation, we first compared the transcriptome profile induced by it in epithelial cells with fibroblast cells. Areca nut was found to induce differential response in these two cell types which corroborates with the disease pathology wherein; epithelial atrophy is observed and conversely fibroblasts are proliferative. To extend these observations we compared the areca nut induced profile in epithelial cells with OSF differential profile and found that a majority of the genes regulated by areca nut which were common with OSF are regulated by TGF-β. Similarly, areca nut and TGF-β regulated profile in fibroblast cells overlapped significantly with OSF profile. Additionally, areca nut and TGF-β treatment positively enriched matrix associated and metabolic pathways among others which are reported to be differentially regulated in OSF. These observations also highlighted the importance of combined actions of areca nut and TGF-β in OSF manifestation. To test the physiological importance of combined actions of areca nut and TGF-β in the context of OSF; activation of fibroblasts by these treatments was assessed. Treatment of fibroblasts with areca nut and TGF-β enhanced the expression of myofibroblast markers αSMA and γSMA with a concomitant increase in the contractile property when compared to areca nut or TGF-β treatment alone. Further, we observed that areca nut did not regulate any of the TGF-β ligands or receptors in fibroblasts, whereas it induced TGF-β2 in epithelial cells. Therefore, this invoked a possible epithelial-mesenchymal interaction that may exist in OSF pathogenesis. To test this possibility in-vitro, epithelial cells were treated with areca nut and the secretome of these cells was put on hGF cells to study the regulation of fibrosis associated genes. This treatment enhanced the regulation of fibroblast activation markers (αSMA and γSMA) as compared to direct areca nut treatment. This increase in regulation was abrogated when induction of TGF-β2 was compromised in epithelial cells. Similar results were obtained for the regulation of other genes (TGM-2, THBS-1, EDN1, LOXL3, PLOD2, TMEPAI, TGFBI, CTGF, BMP1, LMIK1). Therefore, we concluded that TGF-β which is secreted in response to areca nut by epithelial cells influences fibroblasts in combination with areca nut to enhance fibrosis response. Furthermore, the secretome of untreated epithelial cells was found to down regulate the basal expression of fibrosis related genes in fibroblasts, invoking a role for epithelial secretome in regulating the fibrosis progression. Our data highlighted the importance of TGF-β’s influence on fibroblast response in OSF, but the mechanism for the regulation of this cytokine was not known. Areca nut did not induce TGF-β ligands in fibroblast as discussed above, but previous data from our group had reported areca nut mediated up regulation of TGF-β2 in epithelial cells. Therefore, we further elucidated the mechanistic details for this induction using immortalized keratinocytes (HaCaT and HPL1D) and correlated these in OSF tissues. The kinetics of the induction of TGF-β signaling by areca nut (5 µg/ml) in epithelial cells was established. Areca nut induced TGF-β2 transcript, protein and activated the canonical signaling (pSMAD2/3) at 2 hours post treatment, which persisted till 24 hours. The regulation of TGF-β2 mRNA at 2 hours was dependent on active transcription but was independent of protein translation whereas the activation of signaling (pSMAD2) required both transcription and translation at this time point. This warranted probing for the role of TβR-I in the activation of TGF-β signal by areca nut. A small molecule inhibitor was used to abrogate the kinase activity of TβR-I. Areca nut induced TGF-β2 mRNA at 2 hours even in the presence of TβR-I inhibitor whereas the induction was compromised at 24 hours although the activation of SMAD2 at both 2 and 24 hours was compromised in the presence of TβR-I. This result signified that induction of TGF-β signaling was dependent on the TβR-I activity at early and late time points, but the transcription of the ligand was independent of the receptor activity at early time point. These results indicated the activation of some other pathway by areca nut which could regulate the transcription of TGF-β2 and thereby activate TGF-β signaling in epithelial cells. To explore this possibility, a panel of pathway inhibitors was used and only JNK inhibitor compromised areca nut induced TGF-β2 mRNA and pSMAD2. The results were corroborated by transient knockdown of JNK1 and JNK2. Further, JNK was phosphorylated at 30 minutes to 2 hours by areca nut treatment on epithelial cells. This activation was found to be independent of TβR-I activity. In good correlation, activated JNK1/2 was also detected in OSF tissues and was not detectable in normal tissues. Since JNK activation was found to be a pre-requisite for areca nut induced TGF-β signal activation; we further explored the mechanism of JNK activation by areca nut itself. Areca nut mediated activation of JNK was found to be dependent on muscarinic acid receptor, Ca2+/CAMKII and ROS. Inhibition of these significantly compromised areca nut induced pJNK. In line with this, inhibition of muscarinic acid receptor activity, CAMKII and ROS also abrogated areca nut mediated induction of TGF-β2 mRNA and pSMAD2. The regulation of TGF-β signaling by areca nut in epithelial cells was dependent on transcription, and JNK activity was essential for this. We further sought to explore transcription factors which were regulated by JNK and therefore could possibly induce TGF-β2 promoter activity. ATF2 and c-Jun transcription factors were found to be induced at 30 minutes by areca nut and this up regulation also persisted till 24 hours. Further, activation of both ATF2 and c-Jun was dependent on JNK but independent of TβR-I activity. Moreover, areca nut treatment induced translocation of these phoshorylated transcription factors in the nucleus of epithelial cells. Additionally, pATF2 and p-c-Jun were enriched on TGF-β2 promoter after 2 hours of treatment by areca nut. To investigate the importance of this enrichment and regulation of TGF-β signal activation by areca nut, we transiently knocked down these proteins and studied the regulation of TGF-β2. Areca nut induced TGF-β2 mRNA and pSMAD2 was abrogated upon ATF2 and c-Jun knockdown, implicating JNK mediated activation of ATF2 and c-Jun in areca nut induced TGF- β signaling. To explore the significance of this mechanism in OSF, immunohistochemical staining for pATF2 and p-c-Jun was performed on OSF and normal tissues. Both the transcription factors were found in the nuclei of OSF tissues whereas their expression was not detected in normal tissues. This expression also correlated with the previously reported activation of SMAD2 in OSF tissues by our group. Hence, ATF2 and c-Jun were observed to be important in areca nut mediated TGF-β signaling in OSF. In conclusion, the work described in this thesis provides mechanistic details into OSF etiopathogenesis. Combined actions of areca nut and TGF-β induced a response in fibroblasts akin to OSF. Our results advocate a role for epithelial secreted factors in influencing fibroblast response in both normal and disease (OSF) conditions. Further, importance of TGF-β in OSF has been elucidated in terms of enhancing the fibroblast response to areca nut. We have also elucidated the mechanism for areca nut mediated activation of TGF-β signaling and have identified the contribution of JNK/ATF2/Jun axis in this process. This work can impact the management of oral submucous fibrosis by providing newer targets for treatment.

The main concern of this thesis is with desmoplasia - a process in which excessive connective tissue is deposited in a neoplasm. This is a common phenomenon in neoplasia but one whose mechanisms are poorly understood. To study the process, I used a human malignant melanoma cell line (UCT-Mel 7) that was established in this laboratory and that, when injected into athymic mice, gave rise to tumours that showed a number of interesting features. Firstly, the tumour induced a marked desmoplastic response as evidenced by a high content of hydroxyproline in tumour lysates, intense staining for reticulin in sections of the tumour and infiltration of the tumour by host mesenchymal cells. Secondly, the desmoplasia was associated in UCT-Mel 7-derived tumours with an unusual phasic pattern of growth that was related to the in vitro passage number of the melanoma cells. On occasions, murine tumours developed at the site of inoculation of human tumour cells. I have identified 2 possible mechanisms by which UCT-Mel 7 cells could have induced the desmoplastic response: either the tumour cells could have exerted their effect indirectly, i.e. via macrophages, or they could have stimulated the host's stromal cells directly. UCT-Mel 7 cells were shown to be chemotactic for mouse macrophages and human foreskin fibroblasts were stimulated, in a dose-dependent manner, to synthesize increased amounts of collagen when co-cultured with mouse peritoneal exudate cells. Stimulation could only be effected by direct cell:cell contact; medium conditioned by macrophages was not effective. The amount of stimulation was not dependent on the state of activation of the peritoneal cells nor on the strain of mouse used. Tumour cells were also found to act directly. Co-culture of UCT-Mel 7 cells and fibroblasts resulted in increased collagen synthesis by the fibroblasts. Increased synthesis of the protein was reflected in an increase in the amount of collagen mRNA. UCT-Mel 7 cell stimulated in a dose-dependent manner with an absolute requirement for intimate cell:cell contact with the fibroblasts. DNA synthesis was not required. Dexamethasone, retinoic acid and the tumour promoter, phorbol myristate acetate, had significant primary effects on fibroblast collagen synthesis but did not modify the response to melanoma cells. Indomethacin, however, had a minimal primary effect upon the fibroblasts but significantly augmented the melanoma cell effect. The nature of the stimulatory cell:cell contact is still uncertain. The gap junction inhibitor, α-glycyrrhetinic acid, did not diminish the melanoma cell effect. Preliminary findings suggested that cell-surface proteoglycans may be important. Removal of the proteoglycans with the inhibitor of proteoglycan assembly, 4-methylumbelliferyl-β-D-xyloside, abrogated the melanoma cell:fibroblast interaction. Recombinant basic fibroblast growth factor did. not seem to be involved in the desmoplastic response. It was of incidental interest to note that this compound inhibited fibroblast collagen synthesis in a manner that was augmented by the concomitant addition of heparin. A surprising finding was the production of a potent inhibitor of collagen synthesis by superinduced cells of the mouse macrophage cell line, P388D₁. This inhibitor has not been fully characterised.

Tissue engineering has emerged as a promising alternative to conventional orthopaedic grafting therapies. The general paradigm for this approach, in which phenotype-specific cells and/or bioactive growth factors are integrated into polymeric matrices, has been successfully applied in recent years toward the development of bone, ligament, and cartilage tissues in vitro and in vivo. Despite these advances, an optimal cell source for skeletal tissue repair and regeneration has not been identified. Furthermore, the lack of robust, functional orthopaedic tissue interfaces, such as the bone-ligament enthesis, severely limits the integration and biological performance of engineered tissue substitutes. This works aims to address these limitations by spatially controlling the genetic modification and differentiation of fibroblasts into a mineralizing osteoblastic phenotype within three-dimensional polymeric matrices. The overall objective of this project was to investigate transcription factor-based gene therapy strategies for the differentiation of fibroblasts into a mineralizing cell source for orthopaedic tissue engineering applications. Our central hypothesis was that fibroblasts genetically engineered to express Runx2 via conventional and biomaterial-mediated ex vivo gene transfer approaches will differentiate into a mineralizing osteoblastic phenotype. We have demonstrated that a combination of retroviral Runx2 overexpression and glucocorticoid hormone treatment synergistically induces osteoblastic differentiation and biological mineral deposition in primary dermal fibroblasts cultured in monolayer. We report for the first time that glucocorticoids induce osteoblastic differentiation in this model system by modulating the phosphorylation state of a negative regulatory serine residue (Ser125) on Runx2 through an MKP-1-dependent mechanism. Furthermore, we utilized these Runx2-genetically engineered fibroblasts to create mineralized templates for bone repair in vitro and in vivo. Finally, we engineered a heterogeneous bone-soft tissue interface with a novel biomaterial-mediated gene transfer approach. Overall, these results are significant toward the ultimate goal of regenerating complex, higher-order orthopaedic grafting templates which mimic the cellular and microstructural characteristics of native tissue. Cellular therapies based on primary dermal fibroblasts would be particularly beneficial for patients with a compromised ability to recruit progenitors to the sight of injury as result of traumatic injury, radiation treatment, or osteodegenerative disease.

This study demonstrates the use of an adenovirus vector system to study the effect of a DNA repair gene on untransformed human fibroblasts. The bacteriophage T4 pyrimidine dimer DNA glycosylase (den V) gene has been inserted into the E3 region of human adenovirus type 5. The resulting recombinant virus Ad Den V was determined to be producing correctly initiated RNA from the RSV 3' LTR promoter used in the den V expression cartridge inserted into the virus. The effect of the den V gene product on human fibroblasts 'liras examined by assaying for the percent host cell reactivation (%HCR) of Vag production for UV irradiated Ad Den V in comparison to that for a control virus. It was shown that the %HCR was significantly greater for Ad Den V as compared to the control virus in xeroderma pigmentosum (XP) cells. UV survival of adenovirus in XP cells exhibited a two component nature. Introduction of the den V gene into XP group A cells increased the D0 value of the first component of the viral survival curve to a level similar to that of XPC cells, which showed no change in this component irrespective of the presence of the den V gene. It has been suggested that the den V gene is able to partially complement the deficiency in some XP cells because of its small size, allowing it to gain access to the DNA damage site where as the cellular repair enzyme complex can not. Since XPC cells are proficient in their alteration of DNA secondary structure prior to DNA excision repair, these results are consistant with the hypothesis that the first component of UV viral survival curves reflects the pathway involved in accessing the damaged sites. The manuscript of a paper has been included as an appendix. The work theorizes on the origin of mammalian immune system diversity and bacteriophage lambda, and their possible relationship to prokaryotic DNA repair genes. / Thesis / Master of Science (MS)

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CBL proteins are E3 ubiquitin ligases and adaptor proteins. The mammalian homologs – CBL, CBL-B and CBL-3 show broad tissue expression; accordingly, the CBL proteins play roles in multiple cell types. We have investigated the function of the CBL-B protein in hematopoietic cells and fibroblasts. The causative agent of chronic myeloid leukemia (CML) is BCR-ABL. This oncogenic fusion down-modulates CBL-B protein levels, suggesting that CBL-B regulates either the development or progression of CML. To assess the involvement of CBL-B in CML, bone marrow transduction and transplantation (BMT) studies were performed. Recipients of BCR-ABL-infected CBL-B(-/-) cells succumbed to a CML-like myeloproliferative disease with a longer latency than the wild-type recipients. Peripheral blood white blood cell numbers were reduced, as were splenic weights. Yet despite the reduced leukemic burden, granulocyte numbers were amplified throughout the animals. As well, CBLB(-/-) bone marrow (BM) cells possessed defective BM homing capabilities. From these results we concluded that CBL-B negatively regulates granulopoiesis and that prolonged latency in our CBL-B(-/-) BMT animals was a function of perturbed homing.To develop an in vitro model to study CBL-B function we established mouse embryonic fibroblasts (MEFs) deficient in CBL-B expression. Transduction of the wild-type and CBL-B-deficient MEFs with BCR-ABL did not confer transformation; nevertheless, the role of CBL-B in fibroblasts was evaluated. The CBL-B(-/-) MEFs showed enhanced chemotactic migration toward serum in both Transwell migration and time-lapse video microscopy studies. The biochemical response to serum was extensively evaluated leading to the development of a model. We predict that CBL-B deficiency either: (a) augments GRB2-associated binding protein 2 (GAB2) phosphorylation leading to enhanced extracellular signal-regulated kinase (ERK) and protein kinase B (PKB / Akt) signaling, or (b) alleviates negative control of Vav3 resulting in stimulation of Rho effectors. In either case, our results reveal a negative regulatory role for CBL-B in fibroblast migration. The two studies detailed herein expand our knowledge of CBL-B function. They strongly suggest that CBL-B can modulate granulocyte proliferation and point toward a role for CBL-B in the motility of numerous cell types.

<p>To this day, cancer remains the leading cause of mortality worldwide1. A major contributor to cancer progression and metastasis is tumor angiogenesis. The formation of blood vessels around a tumor is facilitated by the complex interplay between cells in the tumor stroma and the surrounding microenvironment. Understanding this interplay and its dynamic interactions is crucial to identify promising targets for cancer therapy. Current methods in cancer research involve the use of two-dimensional (2D) monolayer cell culture. However, cell-cell and cell-ECM interactions that are important in vascularization and the three-dimensional (3D) tumor microenvironment cannot accurately be recapitulated in 2D. To obtain more biologically relevant information, it is essential to mimic the tumor microenvironment in a 3D culture system. To this end, we demonstrate the utility of poly(ethylene glycol) diacrylate (PEGDA) hydrogels modified for cell-mediated degradability and cell-adhesion to explore, in 3D, the effect of various tumor microenvironmental features such as cell-cell and cell-ECM interactions, and dimensionality on tumor vascularization and cancer cell phenotype. </p><p>In aim 1, PEG hydrogels were utilized to evaluate the effect of cells in the tumor stroma, specifically cancer associated fibroblasts (CAFs), on endothelial cells (ECs) and tumor vascularization. CAFs comprise a majority of the cells in the tumor stroma and secrete factors that may influence other cells in the vicinity such as ECs to promote the organization and formation of blood vessels. To investigate this theory, CAFs were isolated from tumors and co-cultured with HUVECs in PEG hydrogels. CAFs co-cultured with ECs organized into vessel-like structures as early as 7 days and were different in vessel morphology and density from co-cultures with normal lung fibroblasts. In contrast to normal lung fibroblasts (LF), CAFs and ECs organized into vessel-like networks that were structurally similar to vessels found in tumors. This work provides insight on the complex crosstalk between cells in the tumor stroma and their effect on tumor angiogenesis. Controlling this complex crosstalk can provide means for developing new therapies to treat cancer.</p><p>In aim 2, degradable PEG hydrogels were utilized to explore how extracellular matrix derived peptides modulate vessel formation and angiogenesis. Specifically, integrin-binding motifs derived from laminin such as IKVAV, a peptide derived from the α-chain of laminin and YIGSR, a peptide found in a cysteine-rich site of the laminin β chain, were examined along with RGDS. These peptides were conjugated to heterobifunctional PEG chains and covalently incorporated in hydrogels. The EC tubule formation in vitro and angiogenesis in vivo in response to the laminin-derived motifs were evaluated. </p><p>Based on these previous aims, in aim 3, PEG hydrogels were optimized to function as a 3D lung adenocarcinoma in vitro model with metastasis-prone lung tumor derived CAFs, HUVECs, and human lung adenocarcinoma derived A549 tumor cells. Similar to the complex tumor microenvironment consisting of interacting malignant and non-malignant cells, the PEG-based 3D lung adenocarcinoma model consists of both tumor and stromal cells that interact together to support vessel formation and tumor cell proliferation thereby more closely mimicking the functional properties of the tumor microenvironment. The utility of the PEG-based 3D lung adenocarcinoma model as a cancer drug screening platform is demonstrated with investigating the effects of doxorubicin, semaxanib, and cilengitide on cell apoptosis and proliferation. The results from drug screening studies using the PEG-based 3D in vitro lung adenocarcinoma model correlate with results reported from drug screening studies conducted in vivo. Thus, the PEG-based 3D in vitro lung adenocarcinoma model may serve as a better tool for identifying promising drug candidates than the conventional 2D monolayer culture method.</p> / Dissertation

Tissue engineered scaffolds were constructed to mimic the native extracellular matrix (ECM) and promote cell migration of keratinocytes and fibroblasts. Electrospinning technology was used to fabricate these nano-scale matrices that consist of varying compositions and fiber diameters. The purpose of this study was to examine how average fiber diameter and scaffold composition regulate cell migration. Odyssey infrared scanning evaluated this on a macroscopic level, whereas confocal microscopy focused on a more microscopic approach. The expression of proteases released into the culture media was also examined. The results from this study suggest that fiber diameter increases as a function of electrospinning starting concentration. Altering the composition by adding a basement membrane-like material, Matrigel, does not statistically affect the average fiber diameter. Fibroblast migration is greater on collagen scaffolds than gelatin scaffolds based on surface area measurements. Confocal images illustrate a distinct cell polarity and various cell morphologies of fibroblasts on electrospun collagen scaffolds. Cell-matrix interactions are more prominent on intermediate to large scale fibers. However, cell-cell contacts are more prevalent at the smallest fiber diameters, suggesting that this scaffold acts like or as a two-dimensional surface. The expression of matrix metalloproteases (MMPs), specifically MMP-2 and MMP-9, by fibroblasts during in vivo cell migration assays, suggests that the greatest amount of matrix remodeling is at the two extremes of fiber diameters.

Cyclin Dependent Kinase-2 Associated Protein-1 (CDK2AP1) plays an important role in cell cycle regulation, by inhibiting CDK2 and by targeting it for proteolysis. It is also known to bind the DNA polymerase alpha-primase complex and regulate the initiation step of DNA synthesis. Its overexpression has been shown to inhibit growth, reduce invasion and increase apoptosis in a number of cancer cell lines. In studies in which mouse embryonic stem cells (mESCs) with targeted deletion of the Cdk2ap1 gene were used, Cdk2ap1 was shown to be required for epigenetic silencing of Oct4 during differentiation. The goal of this thesis was to examine the role of CDK2AP1 in somatic cells (primary human dermal fibroblasts (HDFs)) and human embryonic stem cells (hESCs) and specifically assess its impact on proliferation, self-renewal and differentiation. In the first part of this study, using a short-hairpin RNA (shRNA) approach, we investigated the effect of CDK2AP1 downregulation in HDFs. Outcomes indicated: (a) reduced proliferation, (b) premature senescence, (c) cell cycle alterations, (d) DNA damage, and (e) an increase in p53, p21, and the p53-responsive apoptotic genes BAX and PUMA. Simultaneous downregulation of p53 and CDK2AP1 in HDFs confirmed that observed phenotype was p53 dependent. In the second part of this study, using a shRNA approach, we investigated the role of CDK2AP1 on hESC fate associated with self-renewal and differentiation. We found that CDK2AP1 knockdown in hESCs resulted in: (a) reduced self-renewal (b) enhanced differentiation (c) cell cycle alterations and (d) increase in p53 expression. Results indicate that the knockdown of CDK2AP1 in hESCs enhances differentiation and favors it over a self-renewal fate. Thus, this study has successfully identified novel functions for CDK2AP1, as its knockdown has a significant impact on self-renewal, differentiation and senescence. Results obtained from this study could contribute to development of directed differentiation strategies for generating uniform populations of differentiated phenotypes from hESCs for clinical applications.